Certain proteins with potential commercial uses can require post-translational modifications that are efficiently produced by mammalian cells. However, mammalian cells, such as the industry standard Chinese Hamster Cells (CHO), can be difficult to grow under GMP conditions and require immense resources to propagate at the scale needed for commercial purposes. Animal based bioreactors systems are an attractive alternative to CHO and other mammalian cell based systems due to reasons which include low cost, low maintenance and ease of scalability. However, the post-translational modification of therapeutic proteins, in particular glycosylation, is executed differently in certain animals and plants as compared to mammalian cells such as CHO cells. Transgenic avians, in part because of their prolific egg laying and protein production abilities, have been successfully employed as therapeutic protein bioreactors. In some instances, sugar molecules (i.e., oligosaccharide or glycosylation structures) attached to proteins produced in the oviduct of avians such as chickens and deposited into eggs have been found to have basic structure similar to CHO and human proteins. However, there are some structural carbohydrate elements that are not present on certain proteins produced in the oviduct that can be important for bioactivity and bioavailability in human patients.
The egg white is formed around the yolk as it traverses the oviduct, the avian equivalent of the mammalian fallopian tube. The region of the oviduct in which egg white formation happens is called the magnum and is populated by cells called tubular gland cells (TGCs) which specialize in the synthesis and secretion of egg white proteins.
The two primary classes of glycosylation structures found on proteins, N- and O-linked oligosaccharides, are synthesized by different sets of enzymes. For O-linked oligosaccharides (also referred to as O-glycans) produced in the magnum of laying hens and deposited in the egg white, the enzymatic machinery for oligosaccharide production appears to be similar to that for human O-glycan production, since essentially the same sugars and linkages are present in oligosaccharide structures produced in both humans and in the avian oviduct.
Hen egg white N-linked oligosaccharides (also referred to as N-glycans) have a structure somewhat similar to those found in humans but are typically lacking the terminal galactose and sialic acid sugars. For certain therapeutic proteins, having the terminal galactose and sialic acid can be important for bioavailability and thus efficacy in patients.
Terminal sialic acid residues, which are rarely present or not present at all on N-glycan structures produced in the hen oviduct, shields the N-glycan from recognition by various lectins (receptors that recognize sugar molecules). Proteins with terminal Gal can be bound by lectins expressed in the liver and cleared from the blood circulation in patients (Ashwell and Morell. Adv Enzymol Relat Areas Mol Biol 41: 99-128, 1974). Proteins with the N-glycan having terminal N-acetylglucosamine (GlcNAc), as is typically the case in proteins produced in the hen oviduct, or mannose are bound by lectins expressed on macrophages, also leading to clearance (Schlesinger, et al. Biochem J 192: 597-606, 1980). These results can lead to proteins having a short half-life which often reduces efficacy.
Interestingly, N-glycans produced in other organs in the chicken such as those found in the blood are typically terminated with Gal and/or sialic acid (Ito, et al. Rapid Commun Mass Spectrom 20: 3557-65, 2006; Raju, et al. Glycobiology 10: 477-86, 2000). Thus it is apparent that the chicken genome contains genes that encode all of the enzymes needed to synthesize a fully sialylated N-glycan.
For chicken egg white derived N-glycans, a small percentage of the branches are occupied by Gal and a small percentage of those Gals are capped with sialic acid. For the egg white O-glycans, a high percentage of branches are capped by sialic acid. There is a substantial amount of galactose and sialic acid in egg white proteins, predominantly due to the abundance of O-glycan modified egg white proteins (Feeney, et al. J Biol Chem 235: 2633-7, 1960; Feeney, et al. J Biol Chem 235: 2307-11, 1960; Robinson and Monsey. Biochem J 147: 55-62, 1975). N- and O-glycan synthesis pathways share the same pools of Gal and sialic acid (Varki, et al., Essentials of Glycobiology. Plainview, N.Y., Cold Spring Harbor Laboratory Press, 1999). Thus the levels of Gal and sialic acid that are available for glycan synthesis in TGCs are high and should not be a limiting factor.
The structure of the egg white N-glycans in addition to what is known about the relevant enzymes in mammals gives clues as to the cellular mechanisms that give rise to the egg white N-glycan structures. In mammals, N-glycan synthesis begins in the endoplasmic reticulum with the synthesis of the dolichol oligosaccharide precursor which includes two GlcNAc residues and a number of mannose and glucose residues. This complex is attached to the asparagine of the target protein. The precursor is trimmed back to 3 mannose and 2 GlcNac residues by various glycosidases (termed the core pentasaccharide). GlcNac, Gal and sialic acid residues are then sequentially added by glycosyltransferases. It is at this stage that the diversity of N-glycan structures becomes prominent possibly due to the intracellular levels of the various glycosyltransferases and competition between the glycosyltransferases for free acceptor sites on the growing N-glycan branches (Varki, et al. Essentials of Glycobiology. Plainview, N.Y., Cold Spring Harbor Laboratory Press, 1999).
Starting with GlcNac, there are at least six N-acetylglucosaminyltransferases (GnTs) responsible for the addition of GlcNAc to the trimannosyl core of N-glycans. The high level of branching of egg white N-glycans indicates that all six GnTs may be expressed in oviduct cells of the hen to some extent.
The galactosyltransferases (e.g., (β1,4 galactosyltransferases), referred to as GalTs herein, are a family of at least 7 members which have distinct as well as overlapping roles in the formation of N- and O-glycans. Galactosyltransferase type 1 (GalT1) is thought to be primarily responsible for addition of Gal to the GlcNac residues of all linkages on the N-glycan (Lee, et al., J Biol Chem 276: 13924-34, 2001). The other members of the family, in particular types 2 and 3, are thought to be able to catalyze this transfer though their actual role in N-glycan synthesis appears to be minor. GalT1 is typically expressed in a ubiquitous manner in all cell types, though the levels can vary.
The sialyltransferase (SialT) family catalyzes addition of sialic acid to Gal or N-acetylgalactosamine (GalNac) (in the case of O-linked glycans) as well as other acceptors. With respect to N- and O-glycans, the sialic acid addition is produced by either an α2,3 or α2,6 linkage depending on the specific SialT involved. Human N-glycans can have either or both α2,3 and α2,6 linkages. CHO-produced N-glycans have only the α2,3 linkage, due to a lack of expression of the α2,6 SialTs (Lee, et al. J Biol Chem 264: 13848-55, 1989). Egg white N-glycans and O-glycans also appear to be linked only through the α2,3 linkage.
There are six members of the α2,3 SialT family. Types 1 and 2 may be involved in O-glycan synthesis as they use the Gal-GalNAc chain as an acceptor. Types 3, 4 and 6 apparently can add sialic acid to chains ending in Gal-GlcNac and may be involved in N-glycan and O-glycan synthesis. Type 5 appears to not be involved in O-glycan or N-glycan synthesis but rather may be involved in the addition of sialic acid to ceramide-containing compounds (Harduin-Lepers, et al. Biochimie 83: 727-37, 2001). Very little has been known about the avian α2,3 SialT family other than the expression analysis of type 1 (SialT1) in chick embryos (Kurosawa, et al. Biochim Biophys Acta 1244: 216-22, 1995).
It is currently estimated that the level for Gal at the last (i.e., terminal) or penultimate (i.e., second to last) position in egg white glycans is less than about 10% and the level for terminal sialic acid is less than about 2%. What is needed are birds which produce glycosylated proteins in oviduct tissue, such as magnum tissue, where a greater quantity of galactose and/or sialic acid is added to the N-linked oligosaccharides.